Effect of Sphingosine-1-Phosphate on Intracellular Free Ca2+ in Cat Esophageal Smooth Muscle Cells

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Psi(m) or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 microM. This 1 microM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 microM in the presence of 1 microM cytosolic Ca2+. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

Análisis comparativo de accidentalidad laboral entre el sistema de clasificación de enfermedad y trauma ocupacional del departamento de trabajo de Estados Unidos –OIICS- y el formato único de reporte de accidente de trabajo de Colombia -FURAT- / Comparative analysis of occupational accidents among Occupational Injury and Illness Classification System –OIICS- of the U.S. labor department and the unique format of work accident report of Colombia –FURAT- using American National Standards Institute –ANSI- z-16.1 and z-16.2 standards

El objetivo de este estudio fue describir la accidentalidad laboral en el sector de la construcción en una aseguradora de riesgos laborales (ARL) usando OIICS y el FURAT, y realizar un análisis usando las normas ANSI Z.16.1 y 2 Se trata de un estudio descriptivo en el que se analizaron 340 registros de accidentes seleccionados aleatoriamente. Se clasificaron de acuerdo con las categorías utilizadas en el FURAT y usando la descripción del accidente se clasificó de acuerdo con las categorías de OIICS mediante un software que fue traducido por expertos (profesional bilingüe y médico). Los análisis de las diferentes categorías del sistema y del formato se hicieron cruzando entre dos y tres variables según la metodología ANSI. Según el FURAT, en orden de frecuencia, las lesiones consisten en golpes, contusiones o aplastamientos ocasionados en el ambiente de trabajo y, según su localización, se destacaron los miembros inferiores, las ubicaciones múltiples, la cabeza y los miembros superiores. Según OIICS, las lesiones en los dedos comprometen los músculos, tendones, ligamentos y articulaciones, y son causadas principalmente por armazones, vigas o travesaños, y por martillo; en ojos, por hierros angulares y esquirlas metálicas; en rodillas, por otras piezas y materiales; en manos y dedos por herramientas de mano de golpe y de clavado. Las heridas punzantes son producidas por clavos, puntillas y tachuelas, que afectan principalmente el pie y la planta del pie. La amputación fue ocasionada por materiales metálicos estructurales con pérdida de dedos y uñas. En conclusión, OIICS mostró una ventaja debido al grado de detalle e información descriptiva presentada, lo que, a su vez, permitió que el análisis realizado con ANSI fuera igualmente detallado y específico...

The aim of this study was to describe the occupational accidents in the construction sector in an ARL - Occupational risk insurance- using OIICS and FURAT, and to develop an analysis using ANSI Z.16.1 and Z.16. 2. This is a descriptive study; 340 randomly selected accident records were analyzed. They were classified according to the categories used in the FURAT, and using the accident description they were classified according to the categories of OIICS using software that was translated by experts (bilingual and medical professionals). The analysis of the different categories of the two systems was crossing between two and three categories according to ANSI methodology. According to FURAT, in the nature of the injury, blows, bruises or crushing caused by the work environment are presented in order of frequency. The main affected parts are the lower limbs, in multiple locations, the head and upper limbs. According OIICS, finger injuries compromise muscles, tendons, ligaments and joints. They are caused mainly by shells, beams or crossbars and hammer. The eyes are affected by angular metal and iron splinters; knees by other parts and materials. The hands and fingers are hurt by hand tools and nailing strokes. Puncture wounds are caused by nails, tacks and thumbtacks affecting mainly the foot and the sole. The amputation was caused by structural metallic materials with finger and nail loss. In conclusion, OIICS showed an advantage due to the degree of detail and descriptive information presented, which in turn allowed the analysis carried out with ANSI equally detailed and specific...

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium / 대한마취과학회지

BACKGROUND: Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta. METHODS: Isometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment. RESULTS: Mepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine. CONCLUSIONS: The mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

The Effect of Extrusion Conditions on the Physicochemical Properties and Sensory Characteristics of Millet – Cowpea Based Fura.

A three-factor three level Response surface methodology central composite retortable design (CCRD) was adopted to study the effect of feed composition (X1), feed moisture content (X2) and screw speed (X3) on proximate compositin, amino acid and sensory evaluation during extrusion of pearl millet and cowpea flour mixtures for the purpose of fura production. The mean observed value of protein for the fura extrudates ranged from 11.2 – 16.8%. Analysis of variance indicates that linear and quadratic effects significantly (P<0.05) affected the protein content of fura extrudates as expected. The mean value of lysine for the extrudates ranged from 5.1 - 6.6g/100g protein and the methionine content ranged from 1.3 - 3.8g /100g protein. The regression models fitted to the experimental data showed high coefficients of determinants with R2 = 0.96, 0.94, 0.94, 0.85 and 0.80 for protein (CHON), carbohydrate (CHO), fat (FAT), ash (ASH) and water (HOH) respectively. The R2 = were 0.90, 0.85, 0.86, 0.92, 0.88, 0.85 and 0.93 for lysine, i/leucine, leucine, valine, methionine – cystine, threonine and tryptophan respectively. The coefficients shows good fit. The importance of process variables on system parameters and physical properties could be ranked in the following order: Feed Composition (X1)>Feed Moisture (X2)>Screw Speed (X3). The optimum values obtained for feed composition, feed moisture and screw speed are 36.5% cowpea level, 22.3% feed moisture content and 186.7 rpm respectively. The corresponding optimum values of lysine, protein content, expansion ratio and bulk density are 6.6g/100g protein, 16.6%, 2.8 and 0.52 Kgm-3 respectively. The data obtained from the study could be used for control of product characteristics and possible projection for the commercial production of fura or any enriched protein based food from the blends pearl millet and cowpea.

Effects of Total Alkaloids in Buxus microphylla Leaves on Aorta Smooth Muscle of Rats and Their Mechanisms / 中草药·英文版

Objective To investigate the effects of total alkaloids in Buxus microphylla leaves (ABML) on isolated rats thoracic aorta rings,and then to explore the possible mechanisms underlying the effects.MethodsThoracic aortas of Wistar rats were isolated,removed,and mounted onto an organ bath.The effects of ABML at different concentration on the contraction of isolated thoracic aorta rings (with and without endothelium) precontracted with KC1 or PE were observed with organ bath technique.Dose-effect curves of CaCl2 were recorded by organ bath technique.The concentration of intracellular Ca2+ ([Ca2+]i) increased by PE,KCI,and caffeine in the presence of ABML was determined using Ca2+ sensitive fluorescence indicator Fura-2/AM loaded thoracic aorta vascular smooth muscle (VSM) cells of rats.ResultsIn aorta rings precontracted with PE and KCI,ABML produced concentrationdependent relaxation in both intact and denuded endothelium ring groups.There was no difference in the inhibition of contraction between the intact and denuded endothelium ring groups at the same concentration.Exposure of isolated thoracic aorta rings to ABML led to a significant reduction in the contracting response induced by CaCI2,and shifted the cumulative concentration-response curves to right.ABML could significantly inhibit the extracellular Ca2+ influx induced by PE and KCI under [Ca2+]0 of 1.5 mmol/L,with inhibitory ratios of 40.2％ and 49.9％,respectively.In the case of Ca2+-free,ABML could significantly inhibit the intracellular Ca2+ release induced by PE,with inhibitory ratio of 72.4％.ConclusionABML relaxes thoracic aorta VSM cells by suppressing influx of extracellular Ca2+ via voltage-dependent Ca2+ channel and receptor-operated Ca2+ channel.

M3 Subtype of Muscarinic Receptors Mediate Ca2+ Release from Intracellular Stores in Rat Prostate Neuroendocrine Cells

Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca2+ concentration ([Ca2+]c ) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 micrometer) induced a sharp transient increase in the [Ca2+]c of RPNECs, which was independent of the extracellular Ca2+. In the same RPNECs, high KCl (60 mM), phenylephrine (5micrometer), UTP (P2Y1/2 agonist, 50, micrometer), and alpha, beta-meATP (P2X1/3 agonist, 0.5micrometer) also increased the [Ca2+]c. The ACh-induced [Ca2+]c change (delta[Ca2+]c ) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M3 antagonist, 0.3micrometer), but not by telenzepine (M1 antagonist, 1 micrometer) and himbacine (M2 and M4 antagonist, 1 mircoM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M3 in RPNECs. In summary, RPNECs express M3 muscarinic receptors that are linked to the release of Ca2+ from intracellular stores. The Ca2+ signals of RPNECs might mediate the parasympathetic regulation of prostate gland.

Effects of flunarizine on the intracellular free calcium concentration in acute intracerebral hemorrhage in rats / 第三军医大学学报

Objective To observe the effects of flunarizine on the intracerebral hemorrhage (ICH) of Wistar rats at different time points. Methods An ICH rat model was established by collagenase. The concentrations of intracellular free Ca 2+ at different time points after ICH were determined by the fluorescence detector with an indicator, Fura 2/AM. The effects of flunarizine on the concentrations at different time points were also observed. Results The concentration of intracellular free Ca 2+ began to increase at 0.5 h, increased significantly at 6 h, and peaked at 24 h (about 4 folds as high as that before hemorrhage), but began to decrease at 72 h after acute intracerebral hemorrhage. Flunarizine decreased the concentration of intracellular free Ca 2+ significantly ( P

Galactocerebroside mediated transmembrane Ca2+ signalling in U-87 MG cells: Possible involvement of phosphoinositides.

Antibody to galactocerebroside (anti-GalC) has been shown to evoke a Ca2+ response in cultured glioma U-87 MG cells. The rise in [Ca2+]i was due to release of Ca2+ from the intracellular stores and influx through the plasma membrane. The rise in [Ca2+]i was markedly inhibited by neomycin sulphate and phorbol dibutyrate suggesting the involvement of phosphoinositides in Ca2+ mobilization. The Ca2+ response induced by anti-GalC was rapidly desensitized and repeated addition of anti-GalC did not elevate the [Ca2+]i . Heterologous desensitization was observed with bradykinin and adenosine triphosphate. The intracellular Ca2+ store mobilized by anti-GalC appears to be the IP3 sensitive pool of endoplasmic reticulum. The influx of Ca2+ is mediated by a channel. The Ca2+ influx was also prevented by pretreatment of cells with neomycin sulphate or phorbol dibutyrate. We propose that galactocerebroside may be associated with phospholipase C or other proteins linked to the phosphoinositide pathway of transmembrane signalling and anti-GalC activates the breakdown of phosphoinositides and thus mobilizes Ca2+ in U-87 MG cells.

Changes in Cytosolic Ca2+ Concentration of Single Rabbit Coronary Artery Smooth Muscle Cell during Ischemic Cardioplegic Period

BACKGROUND: No-reflow is a specific type of vascular damage occuring when removal of coronary occlusion dose not lead to restoration of coronary flow. There are three major explanations for the no-reflow phenomenon such as endothelial cell edema, microvascular plugging by platelets or thrombi and coronary occlusion by ischemic contracture of the myocardium. But detailed mechanisms of no-reflow phenomenon are not known. The objects of this study are to elucidate the possibility whether elevation of cytosolic Ca2+ concentration during ischemic cardioplegic period is mechanism of no-reflow phenomenon or not. METHODS: Changes in cytosolic Ca2+ concentration were measured under varying experimental condition. Free [Ca2+] in the cytosole [Ca2+]i of single rabbit coronary artery cells was measured with fluorescent Ca2+ indicator, Fura-2. RESULTS: Resting [Ca2+]i was 134.2+/-34 nM (n=43). When single cells were perfused with cardioplegic or ischemic cardioplegic solution, [Ca2+]i was significantly increased and degree of [Ca2+]i elevation was further augmented by ischemic cardioplegic solution. Pretreatment of sarcoplasmic reticulum emptying agent (20mM caffeine) had no effect on cardioplegia-induced [Ca2+]i change, but application of Ca2+ channel blocker (5x10-7M nifedipine) or an antagonist of Na+/Ca2+ exchange (5mM Ni2+ ) partially (nifedipine) or completely (nickel) inhibited the [Ca2+]i elevation. Pretreament of caffeine had no effect on ischemic cardioplegia-induced [Ca2+]i change, but application of nifedipine or nickel partially inhibited the [Ca2+]i elevation. Magnitude of ischemic cardioplegia-induced [Ca2+]i elevation was dependent on the Ca2+ concentration of perfusate from 0 to 2.5mM. When Ni2+ was added to reperfusion solution, recovery of ischemic cardioplegia-induced [Ca2+]i elevation was very rapid compared with control. CONCLUSIONS: From the above results, it may be speculated that ischemic cardioplegia-induced [Ca2+]i elevation may act as one of the mechanism of no-reflow phenomenon in rabbit coronary artery.

Effect of hypothermic cardioplegia on cardiac protection--I. Effect of hypothermic cardioplegia on the cytosolic Ca2+ concentration in rat ventricular myocytes

Cytosolic Ca2+ concentration of rat ventricular cells was measured under varying experimental conditions by using a fluorescent Ca2+ indicator, Fura-2. Resting [Ca2+]i of rat myocyte was 150 +/- 30 nM (n = 39), and this value was compatible with others. The Perfusion of cardioplegic solution significantly increased [Ca2+]i, and this effect was further augmented by hypothermia (p<0.05). Application of nifedipine (5 x 10(-7) M) to the perfusate or pretreatment of caffeine (10 mM) had no apparent effect on this cardioplegia-induced [Ca2+]i change. But Ni2+ (5 mM), an antagonist of Na+/Ca2+ exchange mechanism, prevented the [Ca2+]i change during cardioplegia (p<0.05). Magnitude of cardioplegia-induced [Ca2+]i increase was also dependent on the Ca2+ concentration of cardioplegic solution. These results suggest that Na+/Ca2+ exchange may play an important role in cardioplegia-induced [Ca2+]i change. To rule out the possibility whether the protective effect of hypothermic cardioplegia is due to the preservation of high-energy phosphate store or decreasing the transmembrane ionic fluxes by phase transition, we exhausted a energy store of cardiac cell by application of 2,4 dinitrophenol to the bath and measured its effect on [Ca2+]i change during cardioplegia. Hypothermic cardioplegia delayed the onset of [Ca2+]i increase and decreased its amplitude compared to those of normothermic cardioplegia. From the above results, hypothermic cardioplegia may protect the cardiac cells from ischemic insult by preserving a high-energy phosphate store. Application of Ni2+ to the cardioplegic solution or reduction of external Ca2+ concentration also had some protective effect, since it prevented [Ca2+]i increase during cardioplegia.

Experimental study of matrine's effects on intracelluar calcium effects on intracell cultured myocardial cells of SD rats / 中成药

Objectives: To clarify the antiarrhythmia mechanism of matrine by observing its effects on intracellular calcium concentration in cultured myocardial cells of SD rats. Methods: Myocardial cells of newborn(sucking) rats were fed with culture medium supplemented with verapamil、 potassium chloride and different dose of matrine, respectively. By means of the image analysis system, intensity changes of intracellular calcium were determined with the fluorescent probe to estimate indirectly inward calcium influx under the influence of matrine. Results: It was found that high potassium caused an increase in fluorescent intensity of intracellular calcium which could be inhibited by matrine. Conclusion: It is suggested that matrine may be a kind of calcium antagonium producing its antiarrhythmic action.

Development of inhibitors of pyrimidine metabolism

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.

Development of inhibitors of pyrimidine metabolism

Benzylacyclouridines were developed as specific and potent competitive inhibitors of uridine phosphorylase with Ki values in the nanomolar range. These compounds have no activity against thymidine phosphorylase, uridine kinase, thymidine kinase and orotate phosphoribosyltransferase. Benzylacyclouridines potentiate the chemotherapeutic effect of FdUrd. Coadministration of uridine phosphorylase inhibitor with FdUrd caused selective toxicity against tumors with low or no thymidine phosphorylase, but not against the host tissues which have thymidine phosphorylase, and thus retain the capacity to cleave FdUrd, and hence overcome its toxicity. There are distinct differences between uridine phosphorylase and thymidine phosphorylase. Benzylacyclouridines competitively inhibit the nucleoside transport of mammalian cells. The structure-activity relationship of inhibitors of uridine phosphorylase showed that a large hydrophobic pocket exists where C-5 of uracil binds, and that it is necessary to have the 3'-hydroxyl group and syn-configuration around the N-glycosidic bond for the nucleosides or their analogs to bind. Dihydrouracil dehydrogenase was found to be widely distributed among mammalian cells, where it was previously believed to be present only in the liver and the kidney. The structure-activity relationship of its inhibitors revealed benzyloxybenzyluracil and 2,6-pyridinediol as most potent. Also identified for orotate phosphoribosyltransferase was 2,4-pyridinediol.

DETERMINATION OF INTERCELLULAR FREE Ca~(2+) CONCENTRATION IN TH E CULTURED CELLS FROM SCHISTOSOMA JAPONICUM BY USING FURA-2/A M / 中国血吸虫病防治杂志

0 05), being ( 187 0?10 7) nmol/L in average. Moreo ver, it was not significantly different from the cells cultured at day 0, ei ther . However, [Ca 2+ ]i increased significantly(P

The effect of ( - )Daurisoline on cytoplasmic free calcium concentration of rabbit lymphocytes / 中国药理学通报

This paper reported the estimation of free calcium concentration in rabbit resting lymphocytes, and the effect of (-) Daurisoline (8701) on the cyto-plasmic free calcium concentration by using fluorescent indicator Fura -2/Am estimating technique. Results were compared with the effect of the Ca2-ionophore A23187.The results indicated that the free calcium concentration of resting lymphocytes was 109. 6nmol ? L-1, after 10-5 mol? L-1,10-4.5 mol ? L-1,and 10-4 mol ? L-1-)Daurisoline administration, the cyto-plasmic free calcium concentration increased to 178. 8%, 204. 4%and 817. 5% respectively, while same concentration A23187 only led the increase of 164. 6% , 198.4% and 327% respectively.Results suggested that ( - )Daurisoline may be a Ca2- agonist and may induce the cytotoxicity effect on lymphocytes at high concentr ation.

Evidence for a major role of Mg~(2+) in bFGF-mediated angiogenesis / 中国药理学通报

Aim The mechanism of basic fibroblast growth factor(bFGF)in mediating increase of intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs), and the relationship between Mg2+and angiogenesis were investigated in this study.Methods The change of[Mg2+]i in HUVECs were quantitatively detected in intracellular cation measurement system via loaded with the fluorescent magnesium indicator mag-fura-2. Endothelial cells were primarily acquired by infusion of collagen enzyme solutioninto the lumens of human umbilical veins and cultured in M199 with 0.2 fetal bovine serum. The role of bFGF in angiogenesis was observed in presence of 0,1 mmol?L-1 or 2 mmol?L-1 of extracellular Mg2+.Results bFGF dose-dependently increased [Mg2+]i, and there was not any significant difference among the groups of 0,1 mmol?L-1and 2 mmol?L-1 of extracellular Mg2+;similar results were obtained in groups done with Na+ and Ca2+. Pretreatment with bFGF receptor-2 (KDR) inhibitor (SU1498) blocked the increase of [Mg2+]i induced by bFGF.Unlike in the group of 0 mmol?L-1extracellular Mg2+,the apparent angiogeneses were observed in the groups of 1 mmol?L-1 and 2 mmol?L-1 extracellular Mg2+ in the presence of bFGF.bFGF-induced angiogenesis was significantly blocked with SU1498 in the presence of 1 mmol?L-1 extracelluar Mg2+.Conclusions These results suggest that the increase of [Mg2+]i by bFGF come from intracellular Mg2+ pools mediated by KDR-dependent signaling pathways,thereby resulting in the bFGF-induced angiogenesis.

The interference of Cd~(2+) with Fura-2 fluorescence in measurement of free cytoplasmic Ca~(2+) / 中国药理学通报

Aim To investigate the blockade of cadmium on store operated calcium channels and the fluorescence interference of cadmium with Fura-2.Methods PC12 cells were used to determine the intracellular calcium concentration [Ca~(2+)]i indicated by change in Fura-2 fluorescence ratio(F_(340)/F_(380)).Results Introduction of cadmium induced a significant increase in Fura-2 fluorescence ratio following thapsigargin-evoked calcium entry;Besides,cadmium gave rise to a remarkable elevation in Fura-2 fluorescence ratio following breakdown of the plasma membrane by triton,a detergent.The Fura-2 fluorescence was increased in the presence of cadmium and Fura-2/AM,simultaneously in the absence of PC12 cells.Conclusion Cadmium can interfere with the Fura-2 fluorescence.

Effects of magnesium salicylate (MgS)on free intracellular calcium in isolated embryonic rat brain cells / 中国药理学通报

AIM To study the calcium antagonistic effects of Magnesium salicylate in central nervous system. MTEHODS Using calcium sensitive fluorescence indicator Fura-2 technique. RESULTS Resting [Ca 2+ ] i in brain cells under different extracellular calcium concentration(0,0 01,0 1,1 0,1 3 mmol?L -1 )were 41?5,59?6,84 7?2 5,104?7,(126?5)nmol?L -1 respectively( n =8～47).MgS 0 2 mmol?L -1 had no significant effects on resting [Ca 2+ ] i.At the same time, MgS (0 05～0 4 mmol?L -1 ) concentration dependently inhibited the [Ca 2+ ] i elevation induced by high extracellular potassium or L -glutamate in the presence of extracellular [Ca 2+ ] i 1 3 mmol?L -1 . The IC 50 values were 0 323 mmol?L -1 (95% CI 0 122～0 854 mmol?L -1 ) and 0 124 mmol?L -1 (95% CI 0 073～0 210 mmol?L -1 ) separately. CONCLUSION MgS possesses inhibitory effects on voltage-activated calcium channel and receptor-operated calcium chanel in CNS.

ANTAGONISTIC EFFECT OF CR1409 ON CHOLECYSTOKININ-INDUCED INCREASE IN CYTOSOLIC CALCIUM CONCENTRATION / 中国药理学通报

The glutaramic acid derivative cholecystokinin ( CCK ) receptor antagonist, CR1409 was tested for its ability to inhibit CCK induced increase in cytosolic calcium concentration in isolated rat pancreatic acinar cells using fura-2 as calcium probe. Preaddition of 10?mol/L CR1409 completely inhibited the increase of cytosolic calcium concertration induced with 1 nmol/L CCK, but had no antagonistic effect on carbacholine bombesin-induced increase in cytosolic calcium concentration. The half maximal inhibition was obtained for 0.37?mol/L, corresponding to a potency a hundred fold higher than that of db cGMP. In the presence of increasing concentrations of CR1409, the response curves of CCK concentrations were shifted to the right but the same maximum was achieved. Schild representation gave a straight line with a slope close to one ( n = 0.92 ) and pA2 value of 6.93. These findings therefore support that CR1409 is a powerful competitive and specific antagonist bf CCK-induced increase in cytosolic calcium Concentration in isolated rat pancreatic acinar cells and provide further evidence for the role of this ion as an intracellular mediator of pancreatic CCK receptor.